AVI BioPharma has been developing an antisense phosphorodiamidate morpholino oligomer, AVI-4020 for the treatment of West Nile Virus. AVI-4020 has been shown both to reduce viral titer, and to cross the blood brain barrier. This compound targets the translation start site of the single open reading frame of WNV. Studies to date indicate AVI-4020 is a reasonable agent for continued clinical development. In order to continue development, more non-clinical studies involving improved understanding of mechanism of action, additional cell culture efficacy and more methodical animal model efficacy studies are necessary. It may be possible to identify an even more potent compound capable of 2 to 3 log greater reduction in viral titer. Hence, this grant is designed to test the feasibility of identification of a more potent agent by making direct comparisons to the existing AVI-4020 agent. If a more potent agent is identified then this will be evaluated to replace AVI-4020 in clinical trials to be proposed in the phase II grant. If no more potent agent is identified then AVI-4020 will have been more rigorously evaluated in non-clinical studies, as is necessary and AVI-4020 will be the agent proposed for clinical evaluation in the phase II grant application. The hypothesis of the study that antisense phosphorodiamidate morpholino oligomer (PMO) antisense agents designed to interfere with RNA-RNA duplex structures in the West Nile Virus (WNV) genome will be more potent the PMOs designed to interfere with initiation of translation of the only WNV ORF. Three specific aims are proposed: Aim 1: Use in vitro viral-reporter constructs to screen for additional viral sites for targeting to identify optimal PMO sequences. Aim 2: Evaluate several candidate PMO inhibitors against whole virus and against reporting replicon constructs in cell culture. Aim 3: Investigate cellular drug transport, protein binding, and physical characteristics, as well as establish a formulation for future animal pharmacokinetic and toxicology studies.